The extracellular matrix (ECM) is a microenvironmental factor that controls cell function. The central theme of this grant proposal is the investigation of the mechanisms by which the ECM controls tumor cell proliferation and how they are affected by proteolytic degradation. We postulate that modification of the ECM by proteases, and in particular matrix metalloproteinases (MMPs), alters the contact between tumor cells and matrix proteins, which subsequently influences the expression of proteins controlling the cell cycle, to favor cell proliferation. In support of this concept we have published observations demonstrating that upon contact with intact fibrillar type I collagen, human M24met melanoma cells are growth arrested at the GI/S interphase and that this arrest involves a specific up-regulation of the cell cyclin dependent kinase inhibitor p27KIP1 (Henriet et al., PNAS, 97:10026-10031, 2000). We have now obtained evidence that up-regulation of p27KIP1 is the consequence of a down-regulation of the S phase kinase associated protein 2 (Skp2) that binds p27KIP1 to ubiquitin conjugase and targets it for degradation by proteasomes. We discovered that down-regulation of Skp2 is dependent on protein kinase Cd (PKCd) activity and have obtained preliminary evidence suggesting that this ECM-dependent regulatory pathway is functional in other melanoma and non-melanoma tumor cell lines. In contrast, in the presence of denatured, non-fibrillar collagen (gelatin), p27KIP1 is down-regulated, cells spread, the focal adhesion kinase protein p125 FAK is phosphorylated, and cells enter into cell cycle. On the basis of these observations, we hypothesize that proteolytic degradation of fibrillar collagen by melanoma cells is necessary to down-regulate p27KIP1, establish functional focal adhesions, and enter the cell cycle, and that preventing collagen deg radation will inhibit cell growth in a p27KIP1/Skp2 dependent manner. Our proposal has 4 specific aims. In the first specific aim we will explore the signaling pathway by which contact with fibrillar collagen activates PKCd and down-regulates Skp2, and obtain proof that p27 and Skp2 are the main regulators downstream of PKCd. In a second specific aim, we will explore the signaling pathway by which contact with denatured and proteolyzed collagen promotes cell proliferation and will determine the role of RhoA, p27KIP1 and Skp2 in this pathway. In the third specific aim, we will examine how proteolytic modification of fibrillar collagen by MMPs changes the environment from growth inhibitory to growth permissive. In a fourth specific aim we will validate our observations in a murine model of melanoma growth and in human melanoma tumor specimens. This information will lead to a better understanding of the role of matrix degrading proteases such as MMPs in cancer, which will be important in the design of better clinical trials involving MMP inhibitors in cancer therapy.